Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.4.1.1 (pyruvate carboxylase)
1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Coryneform bacteria are widely used to produce amino acids, in particularly glutamic acid, by fermentation. To study the metabolic fate of glucose as the carbon source, we developed a method to analyze intracellular extracts by NMR and HPLC. The intracellular metabolites represent the metabolic state of the cells. Glutamic acid was the major metabolic intermediate found in the extracts and its 13C isotopic enrichment reflected that of pyruvic acid. Thus, it was possible to determine the respective contributions of the two major glucose catabolic pathways during the exponential growth phase; glycolysis (55%) and the pentose phosphate pathway (45%). Absolute glutamate 13C enrichments resulting from the incorporation of [1-13C]glucose were determined to quantify the contribution of several metabolic pathways such as anaplerotic pathways (61%; phosphoenolpyruvate carboxylase, pyruvate carboxylase, malic enzyme), a single turn (32%) or multiple turns of the Krebs cycle and the glyoxylate shunt, to oxaloacetate synthesis. A previously described model was adapted to C. melassecola for these calculations. The Krebs cycle was active, whereas the glyoxylate shunt was inactive in exponentially growing cells of C. melassecola with glucose as the sole carbon source. The contributions of anaplerotic enzymes and pyruvate dehydrogenase to replenishing the Krebs' cycle were determined to be 38% and 62%, respectively.
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PMID:13C-NMR studies of Corynebacterium melassecola metabolic pathways. 785 27

A method is presented for determining the compartmentation of amino acid metabolism in the brain. 13C NMR spectroscopy, and more specifically, homonuclear 13C-13C spin coupling patterns of 13C-labeled amino acids were used to measure the relative flux of label from D-[U-13C]glucose through the anaplerotic pathway versus the oxidative pathway. Glucose flux through the pyruvate carboxylase pathway was quantitated following primed dose constant infusion of D-[U-13C]glucose to young rabbits at a rate of 1 mg/kg body weight per min. We demonstrate, for the first time, that multiplet spectra of three adjacent 13C isotopomer in 1,2,3-13C3 in glutamine and glutamate, which are derived from [1,2,3-13C3]pyruvate, present different isotopomer populations in glutamine in comparison to that in glutamate. This is due to two different metabolic compartments characterized by the presence or absence of glutamine synthetase activity and two different tricarboxylic acid cycles, one preferentially mediated by pyruvate carboxylase and the other by pyruvate dehydrogenase. Our results indicate that the anaplerotic pathway accounts for 34% of glutamine synthesis and only 16% of glutamate and gamma-aminobutyric acid syntheses in metabolic and isotopic steady state conditions. These results support the concept, and provide a quantitative measure, that glutamine and/or tricarboxylic acid cycle intermediates are supplied by astrocytes to neurons to replenish the neurotransmitter pool of gamma-aminobutyric acid and glutamate.
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PMID:Cerebral metabolic compartmentation. Estimation of glucose flux via pyruvate carboxylase/pyruvate dehydrogenase by 13C NMR isotopomer analysis of D-[U-13C]glucose metabolites. 796 29

The fate of [3-13C]alanine administered to last instar larvae of an insect Manduca sexta was investigated in vivo by 13C-NMR spectroscopy. Following injection of the isotopically substituted substrate and conversion to [3-13C]pyruvate 13C was principally incorporated into C2, C3 and C4 of glutamate and glutamine in unparasitized ad libitum-fed larvae, insects starved 48 hr prior to injection and larvae parasitized by the insect parasite Cotesia congregata. Selective labeling at C2 and C3 of glutamate/glutamine resulted from carboxylation of [3-13C]pyruvate to [2,3-13C]oxaloacetate catalyzed by pyruvate carboxylase, randomization of the label in fumarate, and synthesis of glutamate and glutamine after condensation with acetyl CoA to [2 proR,3-13C]citrate. In contrast, enrichment at C4 of glutamate and glutamine resulted from oxidation [3-13C]pyruvate to [2-13C]acetyl CoA catalyzed by pyruvate dehydrogenase followed by condensation with oxaloacetate. The ratio of enrichment (C2 + C3): C4 provided a measure of the relative contributions of the pyruvate dehydrogenase and pyruvate carboxylase catalyzed pathways of substrate utilization by the tricarboxylic acid cycle. The mean ratio was 0.6 and 0.7 in control and parasitized larvae, respectively, and 2.4 in starved insects. The latter result demonstrated that substrate utilization by the TCA cycle was markedly altered by starvation. In addition, the rate of labeled alanine metabolism was significantly reduced by starvation. The concentrations of glutamate and glutamine in the blood (hemolymph) were similar in all three groups of insects. No evidence for gluconeogenesis was observed in any group. Starved larvae incorporated label into C6 of glucose and trehalose but no complementary enrichment at C1 was observed. This result was consistent with the activity of the non-oxidative phase of the pentose phosphate pathway during which labeled glyceraldehyde-3-phosphate arising from [3-13C]alanine reacts with sedoheptulose-7-phosphate yielding erythrose-4-phosphate and [6-13C]fructose-6-phosphate catalyzed by transaldolase. Specifically labeled fructose-6-phosphate then gives rise to glucose and trehalose labeled at C6. Preliminary analysis of the hemolymph of starved insects indicated the presence of several hexose phosphates labeled at C6. The hemolymph level of trehalose was significantly reduced in both starved and parasitized insects. Lipogenesis from [3-13C]alanine was evident in unparasitized control larvae but was absent in parasitized and starved insects. The pattern of labeling in fatty acid was consistent with de novo pathway utilizing [2-13C]acetyl CoA derived by oxidation of [3-13C]alanine.
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PMID:Metabolic fate of alanine in an insect Manduca sexta: effects of starvation and parasitism. 810 Jul 13

The metabolism of [1,2-13C2]glucose and [U-13C4]3-hydroxybutyrate was studied in rat brain with in vivo and in vitro 13C NMR spectroscopy, taking advantage, in particular, of homonuclear 13C-13C spin coupling patterns. After infusion of [1,2-13C2]glucose or [U-13C4]3-hydroxybutyrate into rats, the uptake of the substrates in brain and their metabolism to [1-13C]bicarbonate could be detected with in vivo 13C NMR spectroscopy. At the end of the infusion experiment, methanol/HCl/HClO4 extracts of the brain tissue were further analysed by high resolution 13C NMR spectroscopy. The 13C spin coupling patterns revealed entirely different isotopomer distributions for the closely related cerebral metabolites glutamate, glutamine and 4-aminobutyric acid. A quantitative analysis of the 13C spectra demonstrated (i) the existence of two kinetically distinct pools of glutamate, (ii) a pronounced CO2 fixation via pyruvate carboxylase in the glial cells accounting for as much as 38% of the oxaloacetate synthesis in the glial tricarboxylic acid cycle, (iii) a cerebral pyruvate recycling system contributing maximally 17% of the pyruvate metabolism through the pyruvate dehydrogenase in neurons, and (iv) a predominant production of 4-aminobutyric acid from glutamate synthesized in the neurons. In addition, the labelling pattern of N-acetyl aspartate upon infusion of labelled glucose or 3-hydroxybutyrate provided insight into the synthesis of this compound in mammalian brain. While the acetyl moiety originates from the metabolic equivalent of the C-1-C-2 part of cerebral glutamate, the aspartyl moiety is not in direct contact with the intermediates of glycolysis or of the tricarboxylic acid cycles.
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PMID:Cerebral metabolism of [1,2-13C2]glucose and [U-13C4]3-hydroxybutyrate in rat brain as detected by 13C NMR spectroscopy. 810 58

Carbon-13 NMR spectroscopy was used to study the effects of the peroxisome proliferator perfluoro-n-decanoic acid (PFDA) on hepatic carbohydrate metabolism in male Fischer-344 rats. The data indicate that PFDA-treated rats display an inhibition in hepatic [1-13C]glucose and [3-13C]alanine utilization on day 5 posttreatment. PFDA rats show hepatic mean glucose and alanine intensities which are significantly greater (ca. 10-100%) than controls. With [1-13C]-glucose as substrate, PFDA rats show severe to complete inhibition in glycogenesis on days 3 and 5 posttreatment. With [3-13C]alanine as substrate, both groups show functional gluconeogenesis and glycogenesis; however, treated rats show a more transient and less intense C1-glycogen resonance relative to control. These data suggest that PFDA inhibits either the hepatocellular transport of glucose and/or its phosphorylation by glucokinase. The effect of PFDA on TCA cycle activity was determined by monitoring the flow of [3-13C]alanine into glutamate. The relative activity of pyruvate carboxylase (PC) versus pyruvate dehydrogenase (PDH) is represented by the ratio of the glutamate NMR signal intensities (C2 + C3)/C4. PFDA rats show a lower (C2 + C3)/C4 glutamate ratio, suggesting greater relative activity of PDH versus PC in PFDA rats relative to controls. Differences in PDH activity may arise from differences in lipolytic activity. Our data suggest a dysfunction in fatty acid metabolism in PFDA rats and corroborate other studies which show that PFDA inhibits fatty acid oxidation.
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PMID:Effects of the peroxisome proliferator perfluoro-n-decanoic acid on hepatic gluconeogenesis and glycogenesis: a 13C NMR investigation. 815 20

Fatty acids produced a stimulation of gluconeogenesis and either inhibition or no effect on ureagenesis in livers perfused with gluconeogenic substrates and having NH4Cl plus ornithine as the nitrogen source. This finding indicates that stimulation of flux through pyruvate carboxylase is not sufficient to enhance urea production from ammonia. The metabolic action of fatty acids showed the following characteristics: (1) it was concentration-dependent, showing saturation-type kinetics similar to those described for fatty acid oxidation; (2) the stimulatory action on gluconeogenesis was constant and independent of NH4Cl concentration, whereas the inhibition of ureagenesis was variable and dependent on NH4Cl concentration and the degree of reduction of the gluconeogenic substrate; and (3) fatty acids produced apparent reciprocal changes in the state of reduction of the cytosolic and mitochondrial NAD systems. Fatty acid oxidation exerted its effect mainly, if not exclusively, by preventing the gluconeogenic substrate-induced stimulation of ureagenesis. Fatty acids also inhibited ureagenesis without stimulating gluconeogenesis (lactate < 1 mmol/L), ruling out a limiting energy availability as the cause of the inhibition. One or both of the following two mechanisms seem to account for the fatty acid-induced inhibition of ureagenesis from NH4Cl. First, a decreased uptake of ornithine, and second, decreased flux through pyruvate dehydrogenase and probably other NAD(P)-linked mitochondrial dehydrogenases. The correlation found between the ability of fatty acids to inhibit ureagenesis and the state of activation of pyruvate dehydrogenase supports the latter point.
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PMID:Reciprocal changes in gluconeogenesis and ureagenesis induced by fatty acid oxidation. 824 72

Pancreatic islets were cultured for 24 h in the presence of 1 mM glucose, which renders islets incapable of responding to glucose with insulin release. These islets were compared to islets maintained at 20 mM glucose for 24 h. Detritiation of [2-3H]glucose and [5-3H]glucose in 1 mM glucose islets was normal, suggesting that glucose transport and phosphorylation and all enzymes of glycolysis were not down-regulated in the incapacitated islets. 14CO2 formation from [U-14C]glucose and [6-14C]glucose was inhibited up to 80% and 14CO2 from methyl succinate was inhibited up to 60%, indicating that down-regulation at (a) mitochondrial site(s) might explain the incapacitated insulin release. 14CO2 formation from [3,4-14C]glucose (which becomes [1-14C]pyruvate) was decreased, indicating that the reaction catalyzed by pyruvate dehydrogenase was down-regulated. This decrease, however, was not as large as the decreases in 14CO2 formation from [U-14C]glucose, [2-14C]glucose (which becomes [2-14C]pyruvate), or [6-14C]glucose (which becomes [3-14C]pyruvate), indicating that other reactions were also down-regulated. 14CO2 formation from [1-14C]glucose was inhibited less than that from [6-14C]glucose in the incapacitated islets (34 vs 54%) and these rates indicated that flux of glucose through the pentose phosphate pathway was increased in the incapacitated islet, such that 29% (0.4 nmol of 1.4 glucose/100 islets/90 min) was metabolized via this pathway in the incapacitated islet but only 3.4% (0.1 of 2.9 nmol glucose/100 islets/90 min) was metabolized via the pentose pathway in the 20 mM glucose islets. With rates of 14CO2 evolved from glucose labeled at C2 and C6 and from methyl succinate labeled at C1 + C4 and C2 + C3 the 14CO2 ratio formula was used to calculate the ratios of carboxylated and decarboxylated pyruvate. Roughly equal amounts of pyruvate entered the citric acid cycle by each route in islets maintained for 24 h at 1, 5, or 20 mM glucose. The results indicate that decarboxylation and carboxylation of pyruvate were about equally suppressed in incapacitated islets and that direct inhibition of reactions of the cycle was unlikely. This is consistent with evidence which indicates that down-regulation of both pyruvate carboxylase and pyruvate dehydrogenase occurs in incapacitated islets, i.e., under long-term conditions that modify amounts of enzymes (MacDonald et al., 1991, J. Biol. Chem. 266, 22392-22397).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Estimates of glycolysis, pyruvate (de)carboxylation, pentose phosphate pathway, and methyl succinate metabolism in incapacitated pancreatic islets. 837 57

A simple model describing reactions of alanine metabolism in isolated hepatocytes from fasted rats is proposed and applied to radioactive data obtained in experiments in which L-[1-14C]-, L-[2-14C]-, L-[3-14C]-, and L-[U-14C]alanine as well as L-alanine plus NaH14CO3 were used as substrates in parallel. Measurements of the rates of incorporation of the label into glucose and CO2 and of accumulation of [1-14C]pyruvate, [1-14C]lactate, [1-14C]alanine and [1-14C]glutamate plus [1-14C]glutamine from the different substrates used allows to calculate flux of alanine carbon through the various metabolic steps taken into account in the model. The validity of this model is indicated by the agreement found between calculations and measurement of the 14CO2 released from [1-14C]alanine as well as between the values of flux through pyruvate carboxylase calculated in two different ways. It is shown that the oxaloacetate synthesized by pyruvate carboxylase enters into the Krebs cycle and into the pathway of phosphoenolpyruvate synthesis in about equal proportions and that about 40% of the oxaloacetate synthesized as a result of alanine metabolism is derived from the Krebs cycle operation. These results, together with the conclusion that flux of alanine carbon through pyruvate dehydrogenase is negligible, are in agreement with known characteristics of hepatic alanine metabolism in the fasted state and, therefore, provide further evidence for the validity of the model proposed in the present study.
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PMID:A simple model for alanine metabolism in isolated rat hepatocytes. 841 95

Previous work demonstrated that methyl esters of succinate are potent insulin secretagogues in pancreatic islets, while unesterified succinate is not. This can be explained by studies reported here, which show that 14C-labeled dimethyl succinate is metabolized to 14CO2 by pancreatic islets, but that 14C-labeled succinic acid is not metabolized. Islets maintained at 1 mM glucose in tissue culture medium for 1 day lose the ability to release insulin in response to glucose and glucose metabolism is decreased 50-80%. The metabolism of dimethyl [1,4-14C]succinate and dimethyl [2,3-14C]succinate is decreased 50-60% in these incapacitated islets relative to islets maintained at 20 mM glucose. From the ratio of 14CO2 formed from dimethyl [1,4-14C]succinate, relative to that from dimethyl [2,3-14C]succinate, "acetate" ratios of 4.9-6.2 were calculated and from the ratio of 14CO2 formed from [2-14C]glucose, relative to that from [6-14C]glucose, "pyruvate ratios" of 1.6-1.7 were calculated. According to the 14CO2 ratios method, these ratios indicate that 53-66% of pyruvate derived from glucose enters the citric acid cycle via carboxylation and 34-47% enters via decarboxylation. Malic enzyme, which carboxylates pyruvate in the cytosol, was normal in islets maintained at 1 mM glucose. Previous work indicated that inhibition of glucose metabolism in islets maintained at low glucose is due to decreased net synthesis of the mitochondrial enzymes pyruvate dehydrogenase and pyruvate carboxylase [J. Biol. Chem. (1991) 266, 22392-22397], which decarboxylate and carboxylate pyruvate, respectively. Acetate (1 mM) but not pyruvate, when added to islets maintained at low glucose, increased dimethyl succinate metabolism to almost that of islets maintained at high glucose. This is consistent with a low amount of pyruvate dehydrogenase being unable to supply acetyl-CoA for condensation with oxalacetate (derived from succinate) and that the rate of the citric acid cycle could be enhanced by adding acetate which can bypass the reaction catalyzed by pyruvate dehydrogenase.
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PMID:Metabolism of the insulin secretagogue methyl succinate by pancreatic islets. 842 53

The effect of treatment of rats with bacterial endotoxin on gluconeogenesis and the flux through pyruvate kinase, phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase and pyruvate dehydrogenase (PDH) was measured in isolated hepatocytes, prepared from animals starved for 18 h, incubated in the presence of 1 mM pyruvate. The lipopolysaccharide reduced gluconeogenesis by 50% and lowered the flux through pyruvate kinase, PEPCK and pyruvate carboxylase by comparable amounts. There was no effect of endotoxaemia on PDH flux, indicating that the lowered rate of gluconeogenesis is not the result of a redistribution of pyruvate metabolism between oxidation and carboxylation. The results confirm that a stimulation of pyruvate kinase activity following treatment with lipopolysaccharide is not involved in the inhibition of gluconeogenesis, but that the effect resides at the level of phosphoenolpyruvate formation. The most favoured mechanism for the inhibition of glucose synthesis is via an inhibition of PEPCK and subsequent feedback inhibition of pyruvate carboxylase, although a secondary effect at the level of the mitochondria and pyruvate carboxylase cannot be excluded.
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PMID:The effect of treatment of the rat with bacterial endotoxin on gluconeogenesis and pyruvate metabolism in subsequently isolated hepatocytes. 842 54


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